The long term goal of our endeavor is to determine the three dimensional structures of innate immune system receptors involved in host detection of endotoxin, a component of the outer membranes of Gram negative bacteria. Endotoxin (lipopolysaccharide or LPS) is the principal inflammatory component of Gram negative bacteria. Detection of LPS by the host signals infection and is the initial step in activation of defense mechanisms. Determining the three dimensional architecture of these receptors will enable us to understand their function at the molecular level. In this application for an R21 grant we focus on the most uncertain of the steps in x-ray crystallographic structure determination, the crystallization step itself. Although many proteins have now been crystallized and there are certain criteria of purity and homogeneity which must be met, each protein is a new and uncertain endeavor. Two proteins well known to be central to host recognition of LPS are LPS binding protein (LBP) and CD14. We have preliminary data and partial success with LBP and CD14 in that crystals of these proteins have been obtained but they are either too small or too poorly diffracting to be useful. More recently two more proteins have been implicated in host recognition of LPS These are TLR4 and MD-2. Crystallization of these has not yet been attempted. Our work under this grant will be to express multiple forms of these four proteins, study their function to ensure that their function is preserved, and then study their crystallizability.